This form should be used for all taxonomic proposals. Please complete all
those modules that are applicable (and then delete the unwanted sections).
For guidance, see the notes written in blue and the separate document
“Help with completing a taxonomic proposal”
Please try to keep related proposals within a single document; you can copy
the modules to create more than one genus within a new family, for
example.
MODULE 1: TITLE, AUTHORS, etc
Code assigned:
(to be completed by ICTV
officers)
2013.006aV
Short title: Eilat virus, a new species in the genus Alphavirus
(e.g. 6 new species in the genus Zetavirus)
Modules attached
(modules 1 and 9 are required)
1
6
2
7
3
8
4
9
5
Author(s) with e-mail address(es) of the proposer:
Farooq Nasar (corresponding author; [email protected])
Scott C. Weaver (co-corresponding author and member of the ICTV Togaviridae Study Group;
[email protected])
List the ICTV study group(s) that have seen this proposal:
A list of study groups and contacts is provided at
http://www.ictvonline.org/subcommittees.asp . If
in doubt, contact the appropriate subcommittee
chair (fungal, invertebrate, plant, prokaryote or
vertebrate viruses)
Togaviridae Study Group
Nicole C. Arrigo, Chair
[email protected]
ICTV-EC or Study Group comments and response of the proposer:
Date first submitted to ICTV:
Date of this revision (if different to above):
June, 2013
August, 2013
Page 1 of 11
MODULE 2: NEW SPECIES
creating and naming one or more new species.
If more than one, they should be a group of related species belonging to the same genus. All new
species must be placed in a higher taxon. This is usually a genus although it is also permissible for
species to be “unassigned” within a subfamily or family.
Code
2013.006aV
(assigned by ICTV officers)
To create 1 new species within:
Genus: Alphavirus
Subfamily:
Family: Togaviridae
Order: Unassigned
Fill in all that apply.
 If the higher taxon has yet to be
created (in a later module, below) write
“(new)” after its proposed name.
 If no genus is specified, enter
“unassigned” in the genus box.
And name the new species:
GenBank sequence accession
number(s) of reference isolate:
Eilat virus
JX678730.1
Reasons to justify the creation and assignment of the new species:


Explain how the proposed species differ(s) from all existing species.
o If species demarcation criteria (see module 3) have previously been defined for the
genus, explain how the new species meet these criteria.
o If criteria for demarcating species need to be defined (because there will now be more
than one species in the genus), please state the proposed criteria.
Further material in support of this proposal may be presented in the Appendix, Module 9
Page 2 of 11
PROPOSAL OVERVIEW: To designate a new species for Eilat virus within the genus Alphavirus
based on genetic, serologic, and phenotypic data. The name is based on one of the collection sites
during the survey of the Negev desert in Israel during 1982-1984 (1).
Background and overview of justification for creating a new designation for Eilat as a new
alphavirus species:
The genus Alphavirus currently includes 29 species grouped into 10 complexes based on antigenic
and/or genetic similarities (2-5). Most alphaviruses infect terrestrial vertebrates via mosquito-borne
transmission and exhibit a broad host range infecting many different vertebrates including birds,
rodents, equids, humans, and nonhuman primates as well as mosquito species encompassing at least
eight genera (4, 5). Alphaviruses are maintained in endemic transmission cycles and occasionally spill
over into humans and domesticated animals to cause disease. Human infections with Old World
alphaviruses such as Ross River, chikungunya, and Sindbis viruses are typically characterized by fever,
rash, and polyarthritis, whereas infections with the New World alphaviruses Venezuelan, eastern, and
western equine encephalitis viruses can cause fatal encephalitis (4, 5).
Host restricted “insect-only” viruses have not previously been described for alphaviruses, but have
been identified in other arbovirus families. In the family Flaviviridae, Cell-fusing agent, Kamiti River,
Culex fla
, Lammi, and Marisma Mosquito virus have been isolated from mosquitoes
(Aedes spp., Culex spp., Uranotaenia spp. and Ochlerotatus spp.) (6-11). Sigma and Moussa viruses
have been isolated from Drosophila melanogaster and Culex decens, respectively, in the family
Rhabdoviridae (12, 13). Lastly, a new bunyavirus named Gouleako virus was isolated from several
mosquito species (Anopheles spp., Culex spp., and Uranotaenia spp.) (14).
Eilat virus (EILV) was isolated from a pool of Anopheles coustani mosquitoes collected in the Negev
desert of Israel (1). Genetic analysis showed major sequence divergence at both nucleotide and amino
acid levels from other alphaviruses (2). Phylogenetic analyses placed EILV as a sister to the western
equine encephalitis antigenic complex within the main clade of mosquito-borne alphaviruses (2).
Electron microscopy revealed that, like other alphaviruses, EILV virions are spherical, 70 nm in
diameter, and bud from the plasma membrane (2). EILV readily infects a variety of insect cells with
little overt cytopathic effect (2). However, in contrast to typical mosquito-borne alphaviruses, EILV
apparently cannot infect mammalian or avian cell lines (2). EILV is the first mosquito-borne
alphavirus reported to be restricted to insects.
Currently the ICTV definition of a virus species is a “a monophyletic group of viruses whose properties
can be distinguished from those of other species by multiple criteria.” (15). These criteria may include,
but are not limited to, natural and experimental host range, cell and tissue tropism, pathogenicity,
vector specificity, antigenicity, and the degree of relatedness of their genomes or genes (15). Our data
meet many of the criteria as defined by ICTV and support the classification of Eilat virus a new species
within the genus Alphavirus.
Page 3 of 11
Summary of evidence supporting Eilat virus as a new alphavirus species:
1. Genetic, serologic, phylogenetic and phenotypic analyses:
a. Nucleotide and amino acid sequence identity of EILV compared with other
alphaviruses ranges from 57-43% and 58-28%, respectively (Module 9: Appendix,
Table 1).
b. Amino acid comparisons of each individual protein show major sequence divergence
relative to other mosquito-borne viruses. Amino acid divergence for the two most
conserved genes, nsP2 and ns P4, ranges from 35-51% and 23-32%, respectively, and
from 50-58% for E1 and 58-68% for the E2 glycoproteins, respectively (Module 9:
Appendix, Table 2).
c. Complement fixation (CF) and hemagglutination inhibition (HI) assays demonstrate
minimal cross-reactivity with other mosquito-borne alphaviruses (Module 9:
Appendix, Table 3).
d. Neighbor-joining, maximum-likelihood, and Bayesian methods place EILV within the
mosquito-borne clade sister to WEEV complex, with far greater divergence from other
alphaviruses than is observed within any other alphavirus species (Module 9:
Appendix, Figure 1).
e. Electron microscopy reveals that, like other alphaviruses, EILV virions are spherical,
70 nm in diameter, and bud from the plasma membrane of mosquito cells in culture.
(Module 9: Appendix, Figure 2)
f. In contrast to all other mosquito-borne alphaviruses, EILV was unable to infect and
replicate in cell lines representing six vertebrate species, but could readily infect and
replicate in insect cell lines (Module 9: Appendix, Figure 3-5). This is a major
phenotypic and functional difference from all other mosquito-borne alphaviruses and
likely indicates a difference in host range and/or cell/tissue tropism.
g. EILV genomic RNA is incapable of replication in five vertebrate cell lines and the lack
of observed replication is not due to temperature sensitivity or inefficient
electroporation of RNA (Module 9: Appendix, Figure 6-7). This suggests that in
contrast to all other mosquito-borne alphaviruses, EILV host-range is very likely
restricted to insects.
MODULE 9: APPENDIX: supporting material
additional material in support of this proposal
References:
1. Samina I, Margalit J, Peleg J. Isolation of viruses from mosquitoes of the Negev, Israel. Trans R
Soc Trop Med Hyg. 1986;80(3):471-2.
2. Nasar F, Palacios G, Gorchakov RV, Guzman H, Da Rosa AP, Savji N, Popov VL, Sherman MB,
Lipkin WI, Tesh RB, Weaver SC. Eilat virus, a unique alphavirus with host range restricted to
insects by RNA replication. Proc Natl Acad Sci U S A. 2012 Sep 4;109(36):14622-7.
3. Forrester NL, Palacios G, Tesh RB, Savji N, Guzman H, Sherman M, Weaver SC, Lipkin WI.
Genome-scale phylogeny of the alphavirus genus suggests a marine origin. J Virol. 2012
Mar;86(5):2729-38.
Page 4 of 11
additional material in support of this proposal
References:
4. Griffin DE. Alphaviruses, In: Fields B N, Knipe D M, Howley P M, editors. Virology. 5th
edition. New York, N.Y: Lippincott-Raven; Pages 1023-68.
5. Strauss JH, Strauss EG. The alphaviruses: gene expression, replication, and evolution. Microbiol
Rev. 1994 Sep;58(3):491-562.
6. Stollar V, Thomas VL (1975) An agent in the Aedes aegypti cell line (Peleg) which causes fusion
of Aedes albopictus cells. Virology 64:367–377.
7. Crabtree MB, Sang RC, Stollar V, Dunster LM, Miller BR (2003) Genetic and phenotypic
characterization of the newly described insect flavivirus, Kamiti River virus. Arch Virol
148:1095–1118.
8. Kim DY, Guzman H, Bueno R Jr, Dennett JA, Auguste AJ, Carrington CV, Popov VL, Weaver
SC, Beasley DW, Tesh RB. Characterization of Culex Flavivirus (Flaviviridae) strains isolated
from mosquitoes in the United States and Trinidad. Virology. 2009 Mar 30;386(1):154-9.
9. Junglen S, Kopp A, Kurth A, Pauli G, Ellerbrok H, Leendertz FH. A new flavivirus and a new
vector: characterization of a novel flavivirus isolated from uranotaenia mosquitoes from a tropical
rain forest. J Virol. 2009 May;83(9):4462-8.
10. Huhtamo E, Putkuri N, Kurkela S, Manni T, Vaheri A, Vapalahti O, Uzcátegui NY.
Characterization of a novel flavivirus from mosquitoes in northern europe that is related to
mosquito-borne flaviviruses of the tropics. J Virol. 2009 Sep;83(18):9532-40.
11. Vázquez A, Sánchez-Seco MP, Palacios G, Molero F, Reyes N, Ruiz S, Aranda C, Marqués E,
Escosa R, Moreno J, Figuerola J, Tenorio A. Novel flaviviruses detected in different species of
mosquitoes in Spain. Vector Borne Zoonotic Dis. 2012 Mar;12(3):223-9.
12. Longdon B, Obbard DJ, Jiggins FM. Sigma viruses from three species of Drosophila form a
major new clade in the rhabdovirus phylogeny. Proc Biol Sci. 2010 Jan 7;277(1678):35-44.
13. Quan P-L, Junglen S, Tashmukhamedova A, Conlan S, Hutchinson SK, Kurth A, Ellerbrok H,
Egholm M, Briese T, Leendertz FH, Lipkin WI, 2010. Moussa virus: a new member of the
Rhabdoviridae family isolated from Culex decens mosquitoes in Côte d’I
e. V
Re 147:
17-24.
14. Marklewitz M, Handrick S, Grasse W, Kurth A, Lukashev A, Drosten C, Ellerbrok H, Leendertz
FH, Pauli G, Junglen S. Gouleako virus isolated from West African mosquitoes constitutes a
proposed novel genus in the family Bunyaviridae. J Virol. 2011 Sep;85(17):9227-34.
15. http://ictvonline.org/codeOfVirusClassification.asp.
Annex:
Include as much information as necessary to support the proposal, including diagrams comparing the
old and new taxonomic orders. The use of Figures and Tables is strongly recommended but direct
pasting of content from publications will require permission from the copyright holder together with
appropriate acknowledgement as this proposal will be placed on a public web site. For phylogenetic
analysis, try to provide a tree where branch length is related to genetic distance.
Note regarding permission to adapt supporting materials from publications:
The figures and diagrams included in this application have been adapted from American Society of
Microbiology (ASM) publications of which the applicants are authors. The ASM Copyright policy for the
Journal of Virology (http://journals.asm.org/misc/ASM_Author_Statement.dtl) states that authors have
“the ght t republish discrete portions of his/her article in any other publication (including print, CDROM, and other electronic formats) of which he or she is author or editor, provided that proper credit is
given to the original ASM publ c t . “P pe c ed t” me
e the the c py ght l e h w
the t p
Page 5 of 11
f the f t p ge f the PDF e
me l me mbe ye p ge
provided for each figure.
“C py ght © Ame c S c ety f
mbe
d DOI]” f the HTML e
M c b l gy [ e t j
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.” Therefore, proper credit is
Table 1. Comparison of nucleotide and amino acid identity of EILV structural and nonstructural ORFs with other
alphaviruses. Upper diagonal displays percent amino acid identity; lower diagonal contains percent nucleotide
identity. EILV – Eilat Virus; TROV – Trocara virus; AURAV – Aura virus; WHATV – Whataroa virus; SINV –
Sindbis virus; WEEV – Western equine encephalitis virus; EEEV – Eastern equine encephalitis virus; VEEV –
Venezuelan equine encephalitis virus; CHIKV – chikungunya virus; RRV – Ross River virus; UNAV – Una virus;
SFV – Semliki Forest virus; MIDV – Middelburg virus; BFV – Barmah Forest virus; NDUV – Ndumu virus; SESV –
Southern elephant seal virus; SPDV – Salmon pancreas disease virus. Copyright © 2012, Farooq Nasar and Scott C.
Weaver. All Rights Reserved.
Table 2. Comparison of individual EILV proteins with other alphaviruses. Percent amino acid identities are
shown. EILV – Eilat Virus; TROV – Trocara virus; AURAV – Aura virus; WHATV – Whataroa virus; SINV –
Sindbis virus; WEEV – Western equine encephalitis virus; EEEV – Eastern equine encephalitis virus; VEEV –
Venezuelan equine encephalitis virus; CHIKV – chikungunya virus; RRV – Ross River virus; UNAV – Una virus;
SFV – Semliki Forest virus; MIDV – Middelburg virus; BFV – Barmah Forest virus; NDUV – Ndumu virus; SESV –
Southern elephant seal virus; SPDV – Salmon pancreas disease virus. Copyright © 2012, Farooq Nasar and Scott C.
Weaver. All Rights Reserved.
Page 6 of 11
Table 3. Complement fixation (A) and hemagglutination-inhibition (B) tests with Eilat virus and other alphavirus
antigens and hyperimmune mouse ascitic fluids (MIAF). *Reciprocal of heterologous titer/reciprocal of homologous
titer. TROV – Trocara virus; AURAV – Aura virus; WHATV – Whataroa virus; SINV – Sindbis virus; WEEV –
Western equine encephalitis virus; FMV – Ft. Morgan virus; HJV – Highlands J virus; EEEV – Eastern equine
encephalitis virus; VEEV – Venezuelan equine encephalitis virus; EVEV – Everglades virus; MUCV – Mucambo
virus; PIXV – Pixuna virus; MAYV – Mayaro virus; CHIKV – chikungunya virus; ONNV – O’ y g-nyong virus;
RRV – Ross River virus; UNAV – Una virus; SFV – Semliki Forest virus; MIDV – Middelburg virus; BFV –
Barmah Forest virus; NDUV – Ndumu virus. Copyright © 2012, Farooq Nasar and Scott C. Weaver. All Rights
Reserved.
Page 7 of 11
Figure 1. Bayesian phylogenetic tree based on nucleotide sequences of the alphavirus structural ORF. A midpoint
rooted tree is shown with all posterior probabilities <1 shown on major branches. Alphavirus complexes are denoted
in bold and italic. EILV – Eilat Virus; TROV – Trocara virus; AURAV – Aura virus; WHATV – Whataroa virus;
SINV – Sindbis virus; WEEV – Western equine encephalitis virus; EEEV – Eastern equine encephalitis virus; VEEV
– Venezuelan equine encephalitis virus; EVEV – Everglades virus; MUCV – Mucambo virus; PIXV – Pixuna virus;
RNV – Rio Negro virus; MAYV – Mayaro virus; CHIKV – chikungunya virus; RRV – Ross River virus; UNAV –
Una virus; SFV – Semliki Forest virus; MIDV – Middelburg virus; BFV – Barmah Forest virus; NDUV – Ndumu
virus; SESV – Southern elephant seal virus; SPDV – Salmon pancreas disease virus. Copyright © 2012, Farooq Nasar
and Scott C. Weaver. All Rights Reserved.
Figure 2. Eilat virion morphology determined by cryo-electron microscopy and transmission electron microscopy.
20 Å cryo-EM reconstruction of EILV glycoprotein spikes on the virion surface (A). The protrusion possibly
representing the E3 protein is highlighted in purple. SINV glycoprotein spikes are shown as a comparison (147).
Page 8 of 11
EILV virions are shown budding from the surface of C7/10 cells (B). SINV – Sindbis virus; EILV – Eilat virus.
Copyright © 2012, Farooq Nasar and Scott C. Weaver. All Rights Reserved.
Figure 3. Replication kinetics of Eilat virus (EILV) on representative insect (grey, 28°C) (A) and vertebrate
(black, 37°C) (B) cell lines. Monolayers were infected at MOI of 10 PFU/cell. Supernatants were collected at
indicated intervals post-infection and titrated on C7/10 cell monolayers. Each data point represents the mean titer of
samples taken from triplicate infections +/-SD. A6 cells were incubated at 28°C. Copyright © 2012, Farooq Nasar
and Scott C. Weaver. All Rights Reserved.
Figure 4. Replication kinetics of Sindbis virus (SINV) on representative insect (grey, 28°C) (A) and vertebrate
(black, 37°C) (B) cell lines. Monolayers were infected at MOI of 10 PFU/cell. Supernatants were collected at
indicated intervals post-infection and titrated on C7/10 cell monolayers. A6 cells were incubated at 28°C. Hpi –
hours post infection; PFU – plaque forming units. Copyright © 2012, Farooq Nasar and Scott C. Weaver. All Rights
Reserved.
Page 9 of 11
Figure 5. Infection of representative vertebrate (37°C) and insect (28°C) cell lines with EILV-eRFP. 50%
confluent monolayers were infected at MOI of 10 PFU/cell, phase contrast and fluorescent field photographs were
taken at various points post-infection. A6 cells were incubated at 28° C. EILV – Eilat virus; dpi – days post
infection. Copyright © 2012, Farooq Nasar and Scott C. Weaver. All Rights Reserved.
Figure 6. Replication of SINV-eGFP replicon genomic RNA in vertebrate (37° C) and insect (28° C) cell lines.
SINV-eGFP epl c ge m c R A w t
c bed
t
d ≈10 μg l q t f R A we e elect p ted t
vertebrate and insect cells. Phase contrast and fluorescent field photographs were taken at 4 dpe. SINV – Sindbis
virus.
Page 10 of 11
Figure 7. Replication of Eilat virus (EILV) genomic RNA in vertebrate (37° C) and insect (28° C) cell lines. RNA
w t
c bed
t f m the cD A cl e d ≈10 μg l q ts of RNA were electroporated into vertebrate and
insect cells. Phase contrast and fluorescent field photographs were taken at day 4-post-electroporation. Copyright ©
2012, Farooq Nasar and Scott C. Weaver. All Rights Reserved.
Page 11 of 11